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Brain Research 888 (2001) 343–347
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Interactive report
Evidence that neurotensin mediates the central effect of leptin on food
intake in rat q
a,
b
a
Abhiram Sahu *, Robert E. Carraway , Yi-Peng Wang
a
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S-829 Scaife Hall, 3550 Terrace Street Pittsburgh,
PA 15261, USA
b
Department of Physiology, University of Massachusetts Medical School, Worcester, MA 06510, USA
Accepted 27 October 2000
Abstract
Recent evidence suggests that leptin’s action on food intake and body weight regulation is mediated by a number of orexigenic and
anorectic neuronal systems in the hypothalamus. Our previous demonstration that central injections of leptin induce hypothalamic
neurotensin (NT) gene expression in association with a reduced food intake and decreased body weight in rats indicates that NT, an
anorectic peptide, is involved in mediating leptin’s action on feeding and body weight regulation. To begin to examine the relative role of
NT in this regard we evaluated the effects of NT antiserum (NT-AS) or NT receptor antagonist, SR 48692, on the satiety action of leptin
in rats. In the first experiment, 3rd cerebroventricular (i.c.v.) administration of either 1 or 5 ml of NT-AS, 30 min prior to leptin (4 mg)
injection, completely blocked the effects of leptin on food deprivation (FD)-induced feeding. In the second experiment, intraperitoneal
(i.p.) administration of SR 48692 (40 mg / kg) also completely prevented leptin’s satiety action on FD-induced feeding. These results
showing the reversal of leptin’s satiety action by either NT immunoneutralization or NT-receptor antagonism support our hypothesis that
NT is involved in mediating leptin’s action on feeding and further suggest that this neuropeptide is a quantitatively important component
of the leptin sensitive neural circuitry. 2001 Elsevier Science B.V. All rights reserved.
Theme: Neural basis of behavior
Topic: Neuropeptides and behavior
Keywords: Neurotensin; Leptin; SR 48692; Feeding
1. Introduction
Leptin, a product of the ob gene, has been implicated to
play an important role in regulation of a variety of
physiological functions, including food intake and body
weight maintenance [5,10,37]. Although the hypothalamus
has been implicated as the major site of leptin action, the
hypothalamic mechanism(s) by which leptin exerts its
satiety action is not clear. However, several orexigenic and
anorectic neuropeptides and neurotransmitters have been
identified to be potential targets of leptin action in the
q
A part of this work has been presented at the 4th International
Congress of Neuroendocrinology, Kitakyushu, Japan, October 11–16,
1998. Published on the World Wide Web on 1 December 2000.
*Corresponding author. Tel.: 11-412-648-9445; fax: 11-412-3837159.
E-mail address: asahu@pitt.edu (A. Sahu).
hypothalamus [7,15,25,28,30]. In addition, our recent study
suggests that leptin not only alters neuropeptide gene
expression but it may also modulate neuropeptide release
and action [26].
We have recently demonstrated that central leptin administration increased gene expression of neurotensin (NT)
in the hypothalamus in association with decreased food
intake and body weight in the rat [25]. This observation
along with the findings that (1) central injection of NT, a
13 amino acid peptide [3], decreases food intake in a
variety of experimental paradigms [16,17,29]; (2) NT
neurons and terminals are present in those sites of the
hypothalamus that have been implicated in feeding behavior and body weight regulation [8,13,14]; (3) NT gene
expression is decreased in ob /ob mice lacking leptin
[34,35]; and (4) NT neurons in the hypothalamus express
leptin receptor [12] suggest that NT is involved in mediating leptin’s action on feeding and body weight regulation.
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )03107-3
344
A. Sahu et al. / Brain Research 888 (2001) 343 – 347
While our previous study has identified NT as a target of
leptin action, it is not clear whether an increased NT gene
expression [25], and thereby a possible increase in NT
synthesis and release, may play a major role in mediating
the action of leptin on feeding. Thus to examine further the
relative role of NT in mediating leptin action, we examined
the effects of NT antiserum (NT-AS [4,18,33]) and an NT
receptor antagonist, SR 48962 [11], on the satiety action of
leptin in the rat.
2. Materials and methods
2.1. Animals
Adult male Sprague–Dawley rats (Taconic, Germantown, NY), weighing 275–300 g, were housed individually
in a temperature (228C) and light (14 h light and 10 h dark,
lights on 05:00 h) controlled room with free access to food
(rodent chow) and water. After 1 week of acclimatization,
the rats were permanently implanted stereotaxically with
22-gauge stainless steel cannulae (Plastic One, Roanoke,
VA) into the third cerebroventricle under pentobarbital
anesthesia. After 2 weeks of recovery, the animals were
used in the following experiments. Separate groups of rats
were used for different experiments. The rats were handled
daily for 7 days before the experiment in order to minimize
nonspecific stress.
2.2. The effects of NT antiserum ( NT-AS) and NT
receptor antagonist ( SR 48692) on the satiety action of
leptin in rats
(1 ml) on leptin’s (4 mg) satiety action was examined.
Experimental protocol was similar to that of the experiment 1a.
2.2.3. Experiment 2
To assess further the role of endogenous NT in mediating leptin action in the hypothalamus, the effects of a
selective nonpeptide NT-receptor antagonist, SR 48692
[14], on leptin’s satiety action were tested. This experiment
was essentially similar to that of experiment 1a, except that
´ France) was injected i.p. at a
SR 48692 (Sanofi Recherche,
dose of 40 mg / kg or vehicle (0.4 ml) alone prior to i.c.v.
leptin administration. This dose of SR 48692 was selected
on the basis of a previous report [11]. SR 48692 was
dissolved in a drop of Tween 80 and then diluted in sterile
distilled water according to the manufacturer’s instruction.
In a separate study, the effects of SR 48692 on feeding
were compared to that of vehicle-treated groups.
2.3. Statistical analysis
Food intake was expressed as the mean6S.E.M. Statistical analyses were performed with analysis of variance
(ANOVA) followed by Student–Newman–Keuls multiple
range test (GB-STAT Software for the Macintosh, Dynamic Microsystems, Inc., Silver Spring, MD). Comparisons with P,0.05 were considered to be significantly
different.
3. Results
3.1. Response to anti-NT antisera
2.2.1. Experiment 1 a
Rats were injected intracerebroventricularly (i.c.v.) with
5 ml of NT-AS or control rabbit serum (CS) in the middle
of the light period (at |13:00 h when the rats do not eat)
followed 30 min later by 4 mg of rat leptin (rLeptin, R&D
Systems Inc., Minneapolis) in phosphate-buffered saline
(PBS) or PBS alone. Thereafter, food was withdrawn from
all rats. Twenty-two hours later similar injections were
made and 30 min after the last injection preweighed rat
chow was provided to the animals. Cumulative food intake
was monitored for 2-, 4- and 6-h periods. The fasting
paradigm was used to induce feeding during the day time
when the rats do not eat, so that the effects of the drugs
could be easily monitored at certain intervals for a
considerable period. In a separate but similar experiment,
the effect of NT-AS (NT-AS1PBS) on food intake was
compared to that of CS treated animals (CS1PBS).
Concentrations of NT-AS and CS IgG were 4.5 mg / ml.
Preparations of NT-AS and CS, and the specificity of this
antisera have been described previously [4,18,33].
2.2.2. Experiment 1 b
In this experiment, the effects of a small dose of NT-AS
Administration of 4 mg of leptin resulted in a decrease
in cumulative food intake that were 44610% (P50.007),
4469% (P50.007) and 5668% (P50.013) of CS1PBS
control at 2-, 4- and 6-h after injection, respectively (Fig.
1). Prior administration of NT-AS at a dose of 5 ml
completely blocked the effects of leptin on food intake at 4
h (F(1,11)50.11, P50.74 vs. CS1PBS) and 6 h
(F(1,11)50.007, P50.93 vs. CS1PBS). At a 2-h period,
leptin-induced decrease in cumulative food intake was
partially blocked by NT-AS treatment because the food
intake values in leptin1NT-AS group was not significantly
different from either control vehicle (F(1,11)50.27, P5
0.61) or leptin1CS (F(1,11)53.66, P50.08) group.
Administration of NT-AS (5 ml) alone had no effect on
food intake as compared to that of the CS group
(mean6S.E.M. for CS1PBS vs. NT-AS1PBS; 2 h:
4.9660.24 g vs. 4.7660.49 g; 4 h: 5.860.6 g vs.
7.6360.68 g; 6 h: 8.5762.14 g vs. 10.8360.88 g; n53 in
each group).
In the second study, leptin-induced decrease in food
intake at 2-, 4- and 6-h was completely reversed by prior
administration of 1 ml of NT-AS (Fig. 1, right panel; 2 h:
A. Sahu et al. / Brain Research 888 (2001) 343 – 347
345
Fig. 1. Effects of neurotensin antiserum (NT-AS) or control serum (CS) on leptin’s effect on food intake induced by food deprivation. Values are presented
as mean6S.E.M. Numbers in parentheses in this and subsequent figures indicate the number of animals. Values with dissimilar superscripts are significantly
different from each other.
F(1,14)50.30, P50.58 vs. CS1PBS; 4 h: F(1,14)50.05,
P50.81 vs. CS1PBS; 6 h: F(1,14)50.09, P50.76 vs.
CS1PBS). Injection of NT-AS (1 ml) alone had no effect
on food intake (mean6S.E.M. for CS1PBS vs. NT-AS1
PBS; 2 h: 3.9860.45 g vs. 3.7460.44 g; 4 h: 4.9560.8 g
vs. 5.2260.89 g; 6 h: 6.9261.23 g vs. 6.9861.02 g; n57
and 5, respectively).
3.2. Response to SR 48692
Similar to the experiment 1, i.c.v. leptin administration
decreased cumulative food intake to 6168% (P50.02),
4968% (P50.002), and 4367% (P50.0006) of the
vehicle1PBS control at 2-, 4- and 6-h after injection,
respectively (Fig. 2). The effect of leptin on food intake
was completely abolished by prior i.p. administration of
SR 48692 at 2-h (F(1,13)51.549, P50.23 vs. vehicle1
PBS), 4-h (F(1,13)50.468, P50.50 vs. vehicle1PBS),
and 6-h (F(1,13)50.003, P50.95 vs. vehicle1PBS)
periods (Fig. 2). Also the administration of SR 48692
alone had no effect on food intake (values are
mean6S.E.M. for vehicle1PBS vs. SR 486921PBS; 2 h:
4.760.53 g vs. 5.3260.90 g; 4 h: 7.1761.03 g vs.
6.8760.47 g; 6 h: 9.661.85 g vs. 8.7261.15 g, n54 in
each group).
4. Discussion
The present studies demonstrated that intracerebroventricular administration of leptin decreased food intake in
rats previously food deprived for 22 h and that prior
injection of either NT-antibody or NT receptor antagonist,
SR 48692, completely reversed the effects of leptin on
feeding.
Understanding the mechanism by which leptin inhibits
feeding is the subject of intense research. In this regard the
hypothalamus has been implicated as the major site of
leptin action [7,10,25,28]. Initially it was thought that
leptin acts by inhibiting activity of the hypothalamic
neurons producing NPY [30], a potent orexigenic signal
[19,27]. Further studies revealed that sensitivity to leptin
on feeding increased in NPY-knock-out mice [9] suggesting that non-NPY neurons are also involved in mediating leptin action. Subsequently several other hypothalamic
orexigenic neurons have been identified as potential targets
of leptin signaling [25]. Although, amongst the anorectic
signals, melanocortin neuronal system has been identified
as one of the major pathways mediating leptin action
[28,36], accumulating evidence suggests that several other
anorectic signals, including NT, are also the targets of
leptin signaling [5,7,15,25].
The potential involvement of NT in food intake and
346
A. Sahu et al. / Brain Research 888 (2001) 343 – 347
Fig. 2. Effects of neurotensin antagonist, SR 48692, on leptin’s effect on
food intake induced by food deprivation. Values with dissimilar
superscripts are significantly different from each other.
body weight regulation is evident from the findings that (a)
central injection of NT inhibits food intake [16,17,29], (b)
NT gene expression and peptide levels are decreased in
ob /ob mice [34,35], and (c) NT levels in several hypothalamic nuclei are decreased in Zucker obese rats [1]. The
possibility that leptin signaling might involve an effect on
NT neurons was tested in our previous study in which
leptin was shown to increase hypothalamic NT gene
expression [25]. This observation with other supporting
data [34,35] led us to postulate that NT may be one of the
important mediators of leptin action in the hypothalamus.
In the present study, immunoneutralization and receptor
antagonism strategies have been used to address the role of
endogenous NT in mediating leptin’s satiety action in the
hypothalamus. Our finding that prior administration of
either anti-NT antibody or NT-receptor antagonist completely blocked the anorectic action of leptin in FD rats
suggests that NT, released perhaps from hypothalamic
neurons [14], is an essential mediator of leptin’s inhibitory
effect on feeding in rats. Since hypothalamic NT neurons
are known to express leptin receptor [12], it is possible that
leptin exerts a direct action in stimulating NT release.
Recent work suggests that NT and leptin may act synergistically in regulating food intake [2]. In addition, daily
leptin injections for 7 days have been shown to alter
hypothalamic NT content [22]. In total, these results are
consistent with the notion that NT is an important mediator
for leptin and that it may also modulate leptin’s effects on
other neurons involved in feeding behavior.
Finally, it is worth noting that in addition to its own
action on feeding, NT has been shown to regulate some of
the hypothalamic signals that are involved in feeding and
body weight regulation [23]. In this respect, NT and
corticotropin-releasing hormone (CRH) connection is
worth mentioning. The evidence such as NT stimulates
CRH release [24] and NT antagonist, SR 48692, decreases
CRH mRNA levels in the paraventricular nucleus [20],
suggest that NT may have an important role in regulating
CRH neuronal activity. In addition, CRH is a potent
anorectic signal [19,36], and it has also been identified as a
potential target of leptin signaling because (a) leptin
stimulates CRH gene expression [32] and CRH release [6],
and (b) CRH antagonist attenuates leptin’s satiety effect
[32]. Taken together, it is possible that leptin’s effect on
feeding may be mediated through the NT-CRH axis. In
addition, it has been shown recently that NT antagonizes
feeding induced by melanin concentrating hormone
(MCH), an important orexigenic signal of lateral hypothalamic origin [21,31]. We have demonstrated that leptin
inhibits hypothalamic MCH gene expression [25] as well
as MCH-induced food intake in the rat [26]. Thus it is
likely that leptin’s action on CRH and MCH neurons may
also involve the NT neuronal system.
In summary, using immunoneutralization and receptor
antagonism techniques, we have provided new evidence
that reinforces our hypothesis that NT is involved in
mediating leptin action on feeding and further suggests that
this neuropeptide is a quantitatively important component
of the leptin sensitive neural circuitry.
Acknowledgements
This work was supported by Competitive Medical
Research Fund of the University of Pittsburgh Medical
Center and NIH DK52844 to AS. Thanks are due to Ms.
Carolyn M. Phalin for excellent technical assistance and to
Dr. Danielle Gully, Sanofi Recherche, Toulouse Cedex,
France, for supplying SR 48692.
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www.elsevier.com / locate / bres
Interactive report
Evidence that neurotensin mediates the central effect of leptin on food
intake in rat q
a,
b
a
Abhiram Sahu *, Robert E. Carraway , Yi-Peng Wang
a
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S-829 Scaife Hall, 3550 Terrace Street Pittsburgh,
PA 15261, USA
b
Department of Physiology, University of Massachusetts Medical School, Worcester, MA 06510, USA
Accepted 27 October 2000
Abstract
Recent evidence suggests that leptin’s action on food intake and body weight regulation is mediated by a number of orexigenic and
anorectic neuronal systems in the hypothalamus. Our previous demonstration that central injections of leptin induce hypothalamic
neurotensin (NT) gene expression in association with a reduced food intake and decreased body weight in rats indicates that NT, an
anorectic peptide, is involved in mediating leptin’s action on feeding and body weight regulation. To begin to examine the relative role of
NT in this regard we evaluated the effects of NT antiserum (NT-AS) or NT receptor antagonist, SR 48692, on the satiety action of leptin
in rats. In the first experiment, 3rd cerebroventricular (i.c.v.) administration of either 1 or 5 ml of NT-AS, 30 min prior to leptin (4 mg)
injection, completely blocked the effects of leptin on food deprivation (FD)-induced feeding. In the second experiment, intraperitoneal
(i.p.) administration of SR 48692 (40 mg / kg) also completely prevented leptin’s satiety action on FD-induced feeding. These results
showing the reversal of leptin’s satiety action by either NT immunoneutralization or NT-receptor antagonism support our hypothesis that
NT is involved in mediating leptin’s action on feeding and further suggest that this neuropeptide is a quantitatively important component
of the leptin sensitive neural circuitry. 2001 Elsevier Science B.V. All rights reserved.
Theme: Neural basis of behavior
Topic: Neuropeptides and behavior
Keywords: Neurotensin; Leptin; SR 48692; Feeding
1. Introduction
Leptin, a product of the ob gene, has been implicated to
play an important role in regulation of a variety of
physiological functions, including food intake and body
weight maintenance [5,10,37]. Although the hypothalamus
has been implicated as the major site of leptin action, the
hypothalamic mechanism(s) by which leptin exerts its
satiety action is not clear. However, several orexigenic and
anorectic neuropeptides and neurotransmitters have been
identified to be potential targets of leptin action in the
q
A part of this work has been presented at the 4th International
Congress of Neuroendocrinology, Kitakyushu, Japan, October 11–16,
1998. Published on the World Wide Web on 1 December 2000.
*Corresponding author. Tel.: 11-412-648-9445; fax: 11-412-3837159.
E-mail address: asahu@pitt.edu (A. Sahu).
hypothalamus [7,15,25,28,30]. In addition, our recent study
suggests that leptin not only alters neuropeptide gene
expression but it may also modulate neuropeptide release
and action [26].
We have recently demonstrated that central leptin administration increased gene expression of neurotensin (NT)
in the hypothalamus in association with decreased food
intake and body weight in the rat [25]. This observation
along with the findings that (1) central injection of NT, a
13 amino acid peptide [3], decreases food intake in a
variety of experimental paradigms [16,17,29]; (2) NT
neurons and terminals are present in those sites of the
hypothalamus that have been implicated in feeding behavior and body weight regulation [8,13,14]; (3) NT gene
expression is decreased in ob /ob mice lacking leptin
[34,35]; and (4) NT neurons in the hypothalamus express
leptin receptor [12] suggest that NT is involved in mediating leptin’s action on feeding and body weight regulation.
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )03107-3
344
A. Sahu et al. / Brain Research 888 (2001) 343 – 347
While our previous study has identified NT as a target of
leptin action, it is not clear whether an increased NT gene
expression [25], and thereby a possible increase in NT
synthesis and release, may play a major role in mediating
the action of leptin on feeding. Thus to examine further the
relative role of NT in mediating leptin action, we examined
the effects of NT antiserum (NT-AS [4,18,33]) and an NT
receptor antagonist, SR 48962 [11], on the satiety action of
leptin in the rat.
2. Materials and methods
2.1. Animals
Adult male Sprague–Dawley rats (Taconic, Germantown, NY), weighing 275–300 g, were housed individually
in a temperature (228C) and light (14 h light and 10 h dark,
lights on 05:00 h) controlled room with free access to food
(rodent chow) and water. After 1 week of acclimatization,
the rats were permanently implanted stereotaxically with
22-gauge stainless steel cannulae (Plastic One, Roanoke,
VA) into the third cerebroventricle under pentobarbital
anesthesia. After 2 weeks of recovery, the animals were
used in the following experiments. Separate groups of rats
were used for different experiments. The rats were handled
daily for 7 days before the experiment in order to minimize
nonspecific stress.
2.2. The effects of NT antiserum ( NT-AS) and NT
receptor antagonist ( SR 48692) on the satiety action of
leptin in rats
(1 ml) on leptin’s (4 mg) satiety action was examined.
Experimental protocol was similar to that of the experiment 1a.
2.2.3. Experiment 2
To assess further the role of endogenous NT in mediating leptin action in the hypothalamus, the effects of a
selective nonpeptide NT-receptor antagonist, SR 48692
[14], on leptin’s satiety action were tested. This experiment
was essentially similar to that of experiment 1a, except that
´ France) was injected i.p. at a
SR 48692 (Sanofi Recherche,
dose of 40 mg / kg or vehicle (0.4 ml) alone prior to i.c.v.
leptin administration. This dose of SR 48692 was selected
on the basis of a previous report [11]. SR 48692 was
dissolved in a drop of Tween 80 and then diluted in sterile
distilled water according to the manufacturer’s instruction.
In a separate study, the effects of SR 48692 on feeding
were compared to that of vehicle-treated groups.
2.3. Statistical analysis
Food intake was expressed as the mean6S.E.M. Statistical analyses were performed with analysis of variance
(ANOVA) followed by Student–Newman–Keuls multiple
range test (GB-STAT Software for the Macintosh, Dynamic Microsystems, Inc., Silver Spring, MD). Comparisons with P,0.05 were considered to be significantly
different.
3. Results
3.1. Response to anti-NT antisera
2.2.1. Experiment 1 a
Rats were injected intracerebroventricularly (i.c.v.) with
5 ml of NT-AS or control rabbit serum (CS) in the middle
of the light period (at |13:00 h when the rats do not eat)
followed 30 min later by 4 mg of rat leptin (rLeptin, R&D
Systems Inc., Minneapolis) in phosphate-buffered saline
(PBS) or PBS alone. Thereafter, food was withdrawn from
all rats. Twenty-two hours later similar injections were
made and 30 min after the last injection preweighed rat
chow was provided to the animals. Cumulative food intake
was monitored for 2-, 4- and 6-h periods. The fasting
paradigm was used to induce feeding during the day time
when the rats do not eat, so that the effects of the drugs
could be easily monitored at certain intervals for a
considerable period. In a separate but similar experiment,
the effect of NT-AS (NT-AS1PBS) on food intake was
compared to that of CS treated animals (CS1PBS).
Concentrations of NT-AS and CS IgG were 4.5 mg / ml.
Preparations of NT-AS and CS, and the specificity of this
antisera have been described previously [4,18,33].
2.2.2. Experiment 1 b
In this experiment, the effects of a small dose of NT-AS
Administration of 4 mg of leptin resulted in a decrease
in cumulative food intake that were 44610% (P50.007),
4469% (P50.007) and 5668% (P50.013) of CS1PBS
control at 2-, 4- and 6-h after injection, respectively (Fig.
1). Prior administration of NT-AS at a dose of 5 ml
completely blocked the effects of leptin on food intake at 4
h (F(1,11)50.11, P50.74 vs. CS1PBS) and 6 h
(F(1,11)50.007, P50.93 vs. CS1PBS). At a 2-h period,
leptin-induced decrease in cumulative food intake was
partially blocked by NT-AS treatment because the food
intake values in leptin1NT-AS group was not significantly
different from either control vehicle (F(1,11)50.27, P5
0.61) or leptin1CS (F(1,11)53.66, P50.08) group.
Administration of NT-AS (5 ml) alone had no effect on
food intake as compared to that of the CS group
(mean6S.E.M. for CS1PBS vs. NT-AS1PBS; 2 h:
4.9660.24 g vs. 4.7660.49 g; 4 h: 5.860.6 g vs.
7.6360.68 g; 6 h: 8.5762.14 g vs. 10.8360.88 g; n53 in
each group).
In the second study, leptin-induced decrease in food
intake at 2-, 4- and 6-h was completely reversed by prior
administration of 1 ml of NT-AS (Fig. 1, right panel; 2 h:
A. Sahu et al. / Brain Research 888 (2001) 343 – 347
345
Fig. 1. Effects of neurotensin antiserum (NT-AS) or control serum (CS) on leptin’s effect on food intake induced by food deprivation. Values are presented
as mean6S.E.M. Numbers in parentheses in this and subsequent figures indicate the number of animals. Values with dissimilar superscripts are significantly
different from each other.
F(1,14)50.30, P50.58 vs. CS1PBS; 4 h: F(1,14)50.05,
P50.81 vs. CS1PBS; 6 h: F(1,14)50.09, P50.76 vs.
CS1PBS). Injection of NT-AS (1 ml) alone had no effect
on food intake (mean6S.E.M. for CS1PBS vs. NT-AS1
PBS; 2 h: 3.9860.45 g vs. 3.7460.44 g; 4 h: 4.9560.8 g
vs. 5.2260.89 g; 6 h: 6.9261.23 g vs. 6.9861.02 g; n57
and 5, respectively).
3.2. Response to SR 48692
Similar to the experiment 1, i.c.v. leptin administration
decreased cumulative food intake to 6168% (P50.02),
4968% (P50.002), and 4367% (P50.0006) of the
vehicle1PBS control at 2-, 4- and 6-h after injection,
respectively (Fig. 2). The effect of leptin on food intake
was completely abolished by prior i.p. administration of
SR 48692 at 2-h (F(1,13)51.549, P50.23 vs. vehicle1
PBS), 4-h (F(1,13)50.468, P50.50 vs. vehicle1PBS),
and 6-h (F(1,13)50.003, P50.95 vs. vehicle1PBS)
periods (Fig. 2). Also the administration of SR 48692
alone had no effect on food intake (values are
mean6S.E.M. for vehicle1PBS vs. SR 486921PBS; 2 h:
4.760.53 g vs. 5.3260.90 g; 4 h: 7.1761.03 g vs.
6.8760.47 g; 6 h: 9.661.85 g vs. 8.7261.15 g, n54 in
each group).
4. Discussion
The present studies demonstrated that intracerebroventricular administration of leptin decreased food intake in
rats previously food deprived for 22 h and that prior
injection of either NT-antibody or NT receptor antagonist,
SR 48692, completely reversed the effects of leptin on
feeding.
Understanding the mechanism by which leptin inhibits
feeding is the subject of intense research. In this regard the
hypothalamus has been implicated as the major site of
leptin action [7,10,25,28]. Initially it was thought that
leptin acts by inhibiting activity of the hypothalamic
neurons producing NPY [30], a potent orexigenic signal
[19,27]. Further studies revealed that sensitivity to leptin
on feeding increased in NPY-knock-out mice [9] suggesting that non-NPY neurons are also involved in mediating leptin action. Subsequently several other hypothalamic
orexigenic neurons have been identified as potential targets
of leptin signaling [25]. Although, amongst the anorectic
signals, melanocortin neuronal system has been identified
as one of the major pathways mediating leptin action
[28,36], accumulating evidence suggests that several other
anorectic signals, including NT, are also the targets of
leptin signaling [5,7,15,25].
The potential involvement of NT in food intake and
346
A. Sahu et al. / Brain Research 888 (2001) 343 – 347
Fig. 2. Effects of neurotensin antagonist, SR 48692, on leptin’s effect on
food intake induced by food deprivation. Values with dissimilar
superscripts are significantly different from each other.
body weight regulation is evident from the findings that (a)
central injection of NT inhibits food intake [16,17,29], (b)
NT gene expression and peptide levels are decreased in
ob /ob mice [34,35], and (c) NT levels in several hypothalamic nuclei are decreased in Zucker obese rats [1]. The
possibility that leptin signaling might involve an effect on
NT neurons was tested in our previous study in which
leptin was shown to increase hypothalamic NT gene
expression [25]. This observation with other supporting
data [34,35] led us to postulate that NT may be one of the
important mediators of leptin action in the hypothalamus.
In the present study, immunoneutralization and receptor
antagonism strategies have been used to address the role of
endogenous NT in mediating leptin’s satiety action in the
hypothalamus. Our finding that prior administration of
either anti-NT antibody or NT-receptor antagonist completely blocked the anorectic action of leptin in FD rats
suggests that NT, released perhaps from hypothalamic
neurons [14], is an essential mediator of leptin’s inhibitory
effect on feeding in rats. Since hypothalamic NT neurons
are known to express leptin receptor [12], it is possible that
leptin exerts a direct action in stimulating NT release.
Recent work suggests that NT and leptin may act synergistically in regulating food intake [2]. In addition, daily
leptin injections for 7 days have been shown to alter
hypothalamic NT content [22]. In total, these results are
consistent with the notion that NT is an important mediator
for leptin and that it may also modulate leptin’s effects on
other neurons involved in feeding behavior.
Finally, it is worth noting that in addition to its own
action on feeding, NT has been shown to regulate some of
the hypothalamic signals that are involved in feeding and
body weight regulation [23]. In this respect, NT and
corticotropin-releasing hormone (CRH) connection is
worth mentioning. The evidence such as NT stimulates
CRH release [24] and NT antagonist, SR 48692, decreases
CRH mRNA levels in the paraventricular nucleus [20],
suggest that NT may have an important role in regulating
CRH neuronal activity. In addition, CRH is a potent
anorectic signal [19,36], and it has also been identified as a
potential target of leptin signaling because (a) leptin
stimulates CRH gene expression [32] and CRH release [6],
and (b) CRH antagonist attenuates leptin’s satiety effect
[32]. Taken together, it is possible that leptin’s effect on
feeding may be mediated through the NT-CRH axis. In
addition, it has been shown recently that NT antagonizes
feeding induced by melanin concentrating hormone
(MCH), an important orexigenic signal of lateral hypothalamic origin [21,31]. We have demonstrated that leptin
inhibits hypothalamic MCH gene expression [25] as well
as MCH-induced food intake in the rat [26]. Thus it is
likely that leptin’s action on CRH and MCH neurons may
also involve the NT neuronal system.
In summary, using immunoneutralization and receptor
antagonism techniques, we have provided new evidence
that reinforces our hypothesis that NT is involved in
mediating leptin action on feeding and further suggests that
this neuropeptide is a quantitatively important component
of the leptin sensitive neural circuitry.
Acknowledgements
This work was supported by Competitive Medical
Research Fund of the University of Pittsburgh Medical
Center and NIH DK52844 to AS. Thanks are due to Ms.
Carolyn M. Phalin for excellent technical assistance and to
Dr. Danielle Gully, Sanofi Recherche, Toulouse Cedex,
France, for supplying SR 48692.
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